Transcription factors of the NF.kappa.B/Rel family are critical regulators of genes involved in inflammation, cell proliferation and apoptosis (for reviews, see Verma et al., Genes Dev. 9:2723-35, 1995; Siebenlist, Biochim. Biophys. Acta 1332:7-13, 1997; Baeuerle and Henkel, Ann. Rev. Immunol. 12:141-79, 1994; Barnes and Karin, New Engl. J. Med. 336, 1066-71, 1997; Baeuerle and Baltimore, Cell 87:13-20, 1996; Grilli et al., NF-.kappa.B and Rel: Participants in a multiform transcriptional regulatory system (Academic Press, Inc., 1993), vol. 143; Baichwal and Baeuerle, Curr. Biol. 7:94-96, 1997). The prototype member of the family, NF.kappa.B, is composed of a dimer of p50 NF.kappa.B and p65 RelA (Baeuerle and Baltimore, Cell 53:211-17, 1988; Baeuerle and Baltimore, Genes Dev. 3:1689-98, 1989). NF-.kappa.B plays a pivotal role in the highly specific pattern of gene expression observed for immune, inflammatory and acute phase response genes, including interleukin 1, interleukin 8, tumor necrosis factor and certain cell adhesion molecules.
Like other members of the Rel family of transcriptional activators, NF-.kappa.B is sequestered in an inactive form in the cytoplasm of most cell types. A variety of extracellular stimuli including mitogens, cytokines, antigens, stress inducing agents, UV light and viral proteins initiate a signal transduction pathway that ultimately leads to NF-.kappa.B release and activation. Thus, inhibitors and activators of the signal transduction pathway may be used to alter the level of active NF-.kappa.B, and have potential utility in the treatment of diseases associated with NF-.kappa.B activation.
Activation of NF.kappa.B in response to each of these stimuli is controlled by an inhibitory subunit, I.kappa.B, which retains NF.kappa.B in the cytoplasm. I.kappa.B proteins, of which there are six known members, each contain 5-7 ankyrin-like repeats required for association with the NF.kappa.B/Rel dimer and for inhibitory activity (see Beg et al., Genes Dev. 7, 2064-70, 1993; Gilmore and Morin, Trends Genet. 9, 427-33, 1993; Diaz-Meco et al., Mol. Cell. Biol. 13:4770-75, 1993; Haskill et al., Cell 65:1281-89, 1991). I.kappa.B proteins include I.kappa.B.alpha. and I.kappa.B.beta..
NF.kappa.B activation involves the sequential phosphorylation, ubiquitination, and degradation of I.kappa.B. Phosphorylation of I.kappa.B is highly specific for target residues. For example, phosphorylation of the I.kappa.B protein I.kappa.B.alpha. takes place at serine residues S32 and S36, and phosphorylation of I.kappa.B.beta. occurs at serine residues S19 and S23. The choreographed series of modification and degradation steps results in nuclear import of transcriptionally active NF.kappa.B due to the exposure of a nuclear localization signal on NF.kappa.B that was previously masked by I.kappa.B (Beg et al., Genes Dev. 6:1899-1913, 1992). Thus, NF.kappa.B activation is mediated by a signal transduction cascade that includes one or more specific I.kappa.B kinases, a linked series of E1, E2 and E3 ubiquitin enzymes, the 26S proteasome, and the nuclear import machinery. The phosphorylation of I.kappa.B is a critical step in NF-.kappa.B activation, and the identification of an I.kappa.B kinase, as well as proteins that modulate its kinase activity, would further the understanding of the activation process, as well as the development of therapeutic methods.
Several protein kinases have been found to phosphorylate I.kappa.B in vitro, including protein kinase A (Ghosh and Baltimore, Nature 344:678-82, 1990), protein kinase C (Ghosh and Baltimore, Nature 344:678-82, 1990) and double stranded RNA-dependent protein kinase (Kumar et al., Proc. Natl. Acad. Sci. USA 91:6288-92, 1994). Constitutive phosphorylation of I.kappa.B.alpha. by casein kinase II has also been observed (see Barroga et al., Proc. Natl. Acad. Sci. USA 92:7637-41, 1995). None of these kinases, however appear to be responsible for in vivo activation of NF-.kappa.B. For example, phosphorylation of I.kappa.B.alpha. in vitro by protein kinase A and protein kinase C prevent its association with NF-.kappa.B, and phosphorylation by double-stranded RNA-dependent protein kinase results in dissociation of NF-.kappa.B. Neither of these conform to the effect of phosphorylation in vivo, where I.kappa.B.alpha. phosphorylation at S32 and S36 does not result in dissociation from NF-.kappa.B.
Other previously unknown proteins with I.kappa.B kinase activity have been reported, but these proteins also do not appear to be significant activators in vivo. A putative I.kappa.B.alpha. kinase was identified by Kuno et al., J. Biol. Chem. 270:27914-27919, 1995, but that kinase appears to phosphorylate residues in the C-terminal region of I.kappa.B.alpha., rather than the S32 and S36 residues known to be important for in vivo regulation. Diaz-Meco et al., EMBO J 13:2842-2848, 1994 also identified a 50 kD I.kappa.B kinase, with uncharacterized phosphorylation sites. Schouten et al., EMBO J. 16:3133-44, 1997 identified p90.sup.rski as a putative I.kappa.B.alpha. kinase; however, p90.sup.rski is only activated by TPA and phosphorylates I.kappa.B.alpha. only on Ser32, which is insufficient to render I.kappa.B.alpha. a target for ubiquitination. Finally, Chen et al, Cell 84:853-862, 1996 identified a kinase that phosphorylates I.kappa.B.alpha., but that kinase was identified using a non-physiological inducer of I.kappa.B.alpha. kinase activity and requires the addition of exogenous factors for in vitro phosphorylation.
Accordingly, there is a need in the art for an I.kappa.B kinase that possesses the substrate specificity and other properties of the in vivo kinase. There is also a need for improved methods for modulating the activity of proteins involved in activation of NF-.kappa.B, and for treating diseases associated with NF-.kappa.B activation. The present invention fulfills these needs and further provides other related advantages.